Dena B. Dubal

Estradiol protects against ischemic injury.

Clinical studies demonstrate that estrogen replacement therapy in postmenopausal women may enhance cognitive function and reduce neurodegeneration associated with Alzheimers disease and stroke, This study assesses whether physiologic levels of estradiol prevent brain injury in an in vivo model of permanent focal ischemia. Sprague-Dawley rats were ovariectomized; they then were implanted, immediately or at the onset of ischemia, with capsules that produced physiologically low or physiologically high 17β-estradiol levels in serum (10 or 60 pg/mL, respectively), One week after ovariectomy, ischemia was induced. Estradiol pretreatment significantly reduced overall infarct volume compared with oil-pretreated controls (mean ± SD: oil = 241 ± 88; low = 139 ± 91; high = 132 ±88 mm3); this protective effect was regionally specific to the cortex, since no protection was observed in the striatum. Baseline and ischemic regional CBF did not differ between oil and estradiol pretreated rats, as measured by laser Doppler flowmetry. Acute estradiol treatment did not protect against ischemic injury. Our finding that estradiol pretreatment reduces injury demonstrates that physiologic levels of estradiol can protect against neurodegeneration.

Reversing EphB2 depletion rescues cognitive functions in Alzheimer model

Amyloid-β oligomers may cause cognitive deficits in Alzheimer’s disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer’s disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-β-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer’s disease.

Estrogens: Trophic and protective factors in the adult brain

Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Although a thorough understanding of the molecular and cellular mechanisms that underlie this effect requires further investigation, significant progress has been made due to the availability of animal models in which we can test potential candidates. It appears that estradiol can act via mechanisms that require classical intracellular receptors (estrogen receptor alpha or beta) that affect transcription, via mechanisms that include cross-talk between estrogen receptors and second messenger pathways, and/or via mechanisms that may involve membrane receptors or channels. This area of research demands attention since estradiol may be an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Minireview: Neuroprotective Effects of Estrogen—New Insights into Mechanisms of Action

An accumulating body of evidence clearly establishes that estradiol is a potent neuroprotective and neurotrophic factor in the adult: it influences memory and cognition, decreases the risk and delays the onset of neurological diseases such as Alzheimer’s disease, and attenuates the extent of cell death that results from brain injuries such as cerebrovascular stroke and neurotrauma. Thus, estradiol appears to act at two levels: 1) it decreases the risk of disease or injury; and/or 2) it decreases the extent of injury incurred by suppressing the neurotoxic stimulus itself or increasing the resilience of the brain to a given injury. During the past century, the average life span of women has increased dramatically, whereas the time of the menopause has remained essentially constant. Thus, more women will live a larger fraction of their lives in a postmenopausal, hypoestrogenic state than ever before. Clearly, it is critical for us understand the circumstances under which estradiol exerts protective actions a...

Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies

We have shown that 17beta-estradiol exerts profound protective effects against stroke-like ischemic injury in female rats. These effects are evident using physiological levels of estradiol replacement in ovariectomized rats and require hormone treatment prior to the time of injury. The protective actions of estradiol appear to be most prominent in the cerebral cortex, where cell death is not apparent until at least 4 h after the initiation of ischemic injury and where cell death is thought to be apoptotic in nature. Middle-aged rats remain equally responsive to the protective actions of estradiol. The maintenance of responsiveness of the cerebral cortex to the neuroprotective actions of estradiol was unexpected since responsiveness of the hypothalamus to estradiol decreases dramatically by the time animals are middle-aged. We believe that the protective actions of estradiol require the estrogen receptor-alpha, since estradiol does not protect in estrogen receptor-alpha knockout mice. We have also implemented a method of culturing cerebral cortical explants to assess the protective effects of estradiol in vitro. This model exhibits remarkable parallelisms with our in vivo model of brain injury. We have found that 17beta-estradiol decreases the extent of cell death and that this protective effect requires hormone pretreatment. Finally, 17alpha-estradiol, which does not interact effectively with the estrogen receptor, does not protect; and addition of ICI 182,780, an estrogen receptor antagonist, blocks the protective actions of estradiol. We have begun to explore the molecular and cellular mechanisms of estradiol-mediated protection. In summary, our findings demonstrate that estradiol exerts powerful protective effects both in vivo and in vitro and suggest that these actions are mediated by estrogen receptors.

Neuroprotective effects of estradiol in middle-aged female rats

Estrogen replacement therapy in postmenopausal women ameliorates cognitive dysfunction and decreases the risk and/or severity of neurodegenerative conditions such as Alzheimer’s disease and stroke. Furthermore, estradiol exerts neuroprotective effects in a variety of in vitro and in vivo models of brain injury. We have previously shown that physiological levels of estradiol attenuate ischemic brain injury in young female rats. However, neurodegenerative events occur more frequently in elderly women who are chronically hypoestrogenic. Therefore, we investigated whether aging rats remain responsive to the neuroprotective actions of estradiol. Young (3–4 months) and middle-aged (9–12 months) rats were ovariectomized and treated for 1 week with estradiol before middle cerebral artery occlusion (MCAO). Regional cerebral blood flow was monitored in some animals at the time of injury. Brains were collected 24 h after MCAO and infarct volume was analyzed. Our data demonstrate that in both young and aging rats, lo...

Estradiol Attenuates Programmed Cell Death after Stroke-Like Injury

Estradiol is a known neurotrophic and neuroprotective factor. Our previous work demonstrated that replacement with physiological concentrations of estradiol protects the cortex against middle cerebral artery occlusion (MCAO)-induced cell death. The cerebral cortex exhibits caspase-dependent programmed cell death (PCD) in many models of focal cerebral ischemia. We hypothesized that estradiol attenuates PCD during stroke injury. The current study explored the temporospatial pattern of markers of PCD, their relationship to the evolution of injury, and their modulation by estradiol. Rats were ovariectomized and treated with either estradiol or vehicle. One week later, rats underwent MCAO, and brains were collected at 1, 4, 8, 16, and 24 hr. We assessed the temporospatial evolution of infarction volume, DNA fragmentation, and levels of spectrin cleavage products in ischemic cortex. Estradiol led to a delay and attenuation of injury-mediated DNA fragmentation as early as 8 hr after MCAO. Estradiol also dramatically reduced the level of the 120 kDa caspase-mediated spectrin breakdown product (SBDP120) at 4 hr but not at 8 or 16 hr. The SBDP150, produced by caspase and calpain, showed peak levels at 16 hr but was not altered by estradiol. These results strongly suggest that estradiol protects the ischemic cortex by attenuating PCD, thereby reducing caspase activity, DNA fragmentation, and subsequently, overall cell death. These studies deepen our understanding of the mechanisms underlying estrogen-mediated neuroprotection.

Estradiol protects against injury-induced cell death in cortical explant cultures: a role for estrogen receptors.

Estradiol has been shown to exert trophic and protective actions in the brain. Our laboratory has shown that in vivo, low physiological levels of estradiol protect the female rat brain against ischemic injury. In the present study, we used organotypic cortical explant cultures to begin to decipher the mechanisms of estradiols actions. Injury was induced by exposure to kainic acid or potassium cyanide/2-deoxyglucose (KCN/2-DG) for varying lengths of time, and cell death was monitored by LDH release at 2, 6, 12, 24, 48, 72 and 96 h after injury. We found that exposure to 1 mM KCN/2 mM 2-DG for 2 h produced consistent delayed cell death that was detectable by 24 h. The presence of 17beta-estradiol (E2) during the 7 days prior to injury significantly reduced the extent of cell death; whereas, administration of E2 at the time of injury did not protect. The protective effects of estradiol were dose dependent. Low doses of E2 (1, 10, and 30 nM) significantly reduced cell death; however, higher concentrations of E2 (>60 nM) had no protective effect. The observations that low levels of E2 protect against cell death, and that pretreatment is required suggest that the protective actions of estradiol may involve estrogen receptors. Therefore, we examined the ability of 17alpha-estradiol, which does not efficiently activate the estrogen receptor, and the addition of the estrogen receptor antagonist, ICI 182,780, to influence the extent of cell death induced by KCN/2-DG. 17alpha-Estradiol failed to protect, and ICI 182,780 prevented E2 from protecting against cell death. Furthermore, E2 pretreatment is required for more than 24 h to be neuroprotective. Our results clearly show that in cortical explant cultures, estradiol protects cells against ischemic injury, and suggest that these protective actions involve estrogen receptors.

Life Extension Factor Klotho Enhances Cognition

Aging is the primary risk factor for cognitive decline, an emerging health threat to aging societies worldwide. Whether anti-aging factors such as klotho can counteract cognitive decline is unknown. We show that a lifespan-extending variant of the human KLOTHO gene, KL-VS, is associated with enhanced cognition in heterozygous carriers. Because this allele increased klotho levels in serum, we analyzed transgenic mice with systemic overexpression of klotho. They performed better than controls in multiple tests of learning and memory. Elevating klotho in mice also enhanced long-term potentiation, a form of synaptic plasticity, and enriched synaptic GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit with key functions in learning and memory. Blockade of GluN2B abolished klotho-mediated effects. Surprisingly, klotho effects were evident also in young mice and did not correlate with age in humans, suggesting independence from the aging process. Augmenting klotho or its effects may enhance cognition and counteract cognitive deficits at different life stages.

Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

Astrocytes express a variety of G protein–coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimers disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.